Circular RNA was first discovered in 1979 by electron microscope (Hsu et al., Nature (1979) 280:339-340; herein incorporated by reference in its entirety). With its 5′ and 3′ ends joined together, circRNA has no free ends and has extraordinary long half-life (Harland & Misher, Development (1988) 102:837-852; herein incorporated by reference in its entirety). Recent studies have confirmed that circRNA is resistant to digestion with RNase R exonuclease and turns over more slowly than its counterpart linear RNA in vivo (Memczak et al. Nature (2013) 495:333-338; herein incorporated by reference in its entirety). An analysis of circRNA and their associated linear mRNAs revealed that the circRNA isoforms were highly stable, with transcript half-lives exceeding 48 hours, while the associated linear transcripts exhibited half-lives of less than 20 hours (Jeck et al., RNA (2013) 19:141-157; herein incorporated by reference in its entirety).
Since their initial discovery circRNAs have been developed for various uses. In U.S. Pat. No. 5,766,903 to Sarnow et al., herein incorporated by reference in its entirety, circRNAs comprise an internal ribosome entry site (IRES) element that engages a eukaryotic ribosome and an RNA sequence element encoding a polypeptide operatively linked to the IRES. The circRNA described by Sarnow can then be inserted into cells in order to produce a polypeptide of interest. U.S. Pat. No. 5,580,859 to Felgner et al., herein incorporated by reference in its entirety, describes polynucleotide sequences, which may be circularized, which may be administered directly to tissues in order to produce proteins. CircRNAs for vascular disease are described in International Publication No. WO2012050975, herein incorporated by reference in its entirety, where Sharpless et al. described circRNAs comprising one or more ANRIL exons which play an active role in atherosclerotic vascular disease. U.S. Pat. No. 5,426,180 to Kool et al., herein incorporated by reference in its entirety, discloses single-stranded circular oligonucleotides that bind to both single-stranded and double-stranded target nucleic acids.
The production of circRNAs has been attempted by various methods such as the method described in U.S. Pat. No. 6,210,931 to Feldstein et al., herein incorporated by reference in its entirety, which teaches a method of synthesizing circRNAs by inserting DNA fragments into a plasmid containing sequences having the capability of spontaneous cleavage and self-circularization. Another method is described in U.S. Pat. No. 5,773,244 to Ares Jr. et al. which teaches producing circRNAs by making a DNA construct encoding an RNA cyclase ribozyme, expressing the DNA construct as an RNA, and then allowing the RNA to self-splice, which produces a circRNA free from intron in vitro. International Publication No. WO1992001813 to Ruth et al., herein incorporated by reference in its entirety, teaches a process of making single strand circular nucleic acids by synthesizing a linear polynucleotide, combining the linear nucleotide with a complementary linking oligonucleotide under hybridization conditions, and ligating the linear polynucleotide.
However, the synthetic circRNA molecules are still susceptible to the pitfalls of their linear counterparts including, but not limited to, reduced structural and functional integrity and/or triggering bio-responses such as the immune response and/or degradation pathways.
It has been previously shown that certain linear modified mRNA sequences have the potential as therapeutics. Such studies are detailed in International Publication No. WO2012019168, filed Aug. 5, 2011, International Publication No. WO2012045075, filed Oct. 3, 2011, International Publication No. WO2012135805, filed Apr. 2, 2012, International Publication No. WO2012045082, filed Oct. 3, 2011, International Publication No. WO2013052523, filed Oct. 3, 2012, and International Publication No. WO2013090648, filed Dec. 14, 2012, the contents of each of which are herein incorporated by reference in its entirety.
The present invention provides single stranded circular polynucleotides (circP) which may comprise structural and/or chemical features such as, but not limited to, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity, overcoming the threshold of expression, improving expression rates, half-life and/or protein concentrations, optimizing protein localization, and avoiding deleterious bio-responses such as the immune response and/or degradation pathways. The circular polynucleotides which may comprise the structural and/or chemical features described herein may have potential in the fields of therapeutics, diagnostics, reagents and for biological assays.